Fluid mixing and delivery in microfluidic systems

ABSTRACT

The specification generally discloses systems and methods for mixing and delivering fluids in microfluidic systems. The fluids can contain, in some embodiments reagents that can participate in one or more chemical or biological reactions. Some embodiments relate to systems and methods employing one or more vent valves to controllably flow and/or mix portions of fluid within the microfluidic system. Advantageously, fluid control such as a sequence of fluid flow and/or a change in flow rate, can be achieved by opening and closing one or more vent valves and by applying a single source of fluid flow (e.g., a vacuum) operated at a substantially constant pressure. This can simplify the operation and use of the device by an intended user.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No. 61/263,981, filed Nov. 24,2009, which is incorporated herein by reference.

FIELD OF INVENTION

Systems and methods for mixing and delivering fluids in microfluidicsystems are generally described. In some cases, the fluids containreagents that can participate in one or more chemical or biologicalreactions.

BACKGROUND

The manipulation of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various microfluidicmethods and devices, such as microfluidic assays, can provideinexpensive, sensitive and accurate analytical platforms, fluidmanipulations—such as the mixture of multiple fluids, sampleintroduction, introduction of reagents, storage of reagents, separationof fluids, collection of waste, extraction of fluids for off-chipanalysis, and transfer of fluids from one chip to the next—can add alevel of cost and sophistication. Accordingly, advances in the fieldthat could reduce costs, simplify use, and/or improve fluidmanipulations in microfluidic systems would be beneficial.

SUMMARY OF THE INVENTION

Systems and methods for mixing and delivering fluids in microfluidicsystems are generally described. The subject matter of the presentinvention involves, in some cases, interrelated products, alternativesolutions to a particular problem, and/or a plurality of different usesof one or more systems and/or articles.

In one set of embodiments, a series of methods are provided. In oneembodiment, a method comprises providing a device comprising a mainchannel, a first branching channel containing a first fluid, a secondbranching channel containing a second fluid, wherein the first andsecond branching channels connect at an intersection and are fluidicallyconnected to the main channel, and a vent valve positioned between aportion of the first branching channel and a portion of the mainchannel. The method involves actuating the vent valve, causing the firstand second fluids to flow into the intersection substantiallysimultaneously, and mixing at least portions of the first and secondfluids to produce a mixed fluid.

In another embodiment, a method comprises providing a device comprisingan upstream channel portion containing a first fluid, a downstreamchannel portion containing a second fluid different from the firstfluid, and a vent valve positioned between the upstream and downstreamchannel portions. While the first and second channel portions are influid communication with one another, the second fluid is flowed in thedownstream channel portion without substantially flowing the firstfluid. The method also includes flowing the second fluid from theupstream channel portion to the downstream channel portion after theflowing of the first fluid.

In another set of embodiments, a series of devices are provided. In oneembodiment, a device comprises an inlet, an outlet, an upstream channelportion in fluid communication with the inlet, a downstream channelportion in fluid communication with the outlet, and a vent valvepositioned between the downstream and upstream channel portions. A firstfluid is stored in at least one of the upstream and downstream channelportions, and the device is sealed and constructed and arranged forstoring the first fluid in the device for at least one hour prior tofirst use.

In another embodiment, a device comprises an inlet, an outlet, a mainchannel between the inlet and the outlet, and a first and a second ventvalve positioned in series along the main channel between the inlet andthe outlet.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 includes a schematic illustration of a device including aplurality of vent valves, according to one set of embodiments;

FIGS. 2A-2F include, according to one set of embodiments,cross-sectional schematic illustrations of vent valves that can be usedin devices described herein;

FIGS. 3A-3D include exemplary schematic diagrams of channels includingone or more vent valves, according to one set of embodiments;

FIGS. 4A-4I include schematic diagrams of branched channels, accordingto one set of embodiments;

FIGS. 5A-5B include schematic illustrations of fluid plugs in a channelof a device, according to one set of embodiments;

FIGS. 6A-6C include exemplary schematic illustrations of variousarrangements of fluid plugs in channels of a device, according to oneset of embodiments;

FIG. 7 includes an exemplary schematic illustration of a deviceincluding a plurality of detection regions, according to one set ofembodiments; and

FIG. 8 includes a plot of cumulative volume of mixed fluid as a functionof time, according to one set of embodiments.

DETAILED DESCRIPTION

The specification generally discloses systems and methods for mixing anddelivering fluids in microfluidic systems. The fluids can contain, insome embodiments, reagents that can participate in one or more chemicalor biological reactions. Some embodiments relate to systems and methodsemploying one or more vent valves to controllably flow and/or mixportions of fluid within a microfluidic system. The vent valves cancomprise, for example, a port in fluid communication with themicrofluidic channel in which a fluid is positioned, and may be actuatedby positioning a seal over the port opening or by removing the seal fromthe port opening. In certain embodiments, the seal may include a valvingmechanism such as a mechanical valve operatively associated with a tubein fluid communication with the port. Generally, opening the vent valveallows the port to function as a vent. When the port functions as avent, the fluid located on one side of the vent valve flows, while thefluid located on the opposite side of the vent valve relative to thefirst fluid remains stationary. When the valve is closed, the port nolonger functions as a vent, and the fluid located on both sides of thevent valve can flow through the system towards an outlet.Advantageously, fluid control such as a sequence of fluid flow and/or achange in flow rate, can be achieved by opening and closing one or morevent valves and by applying a single source of fluid flow (e.g., avacuum) operated at a substantially constant pressure. This can simplifythe operation and use of the device by an intended user.

Vent valves can be actuated so as to control the movement of fluid inthe microfluidic system. For example, fluids can be stored serially in achannel, and after closing a vent valve positioned along the channel,the fluids can flow sequentially towards the channel outlet. In somecases, fluids can be stored in separate, intersecting channels, andafter closing a vent valve the fluids will flow together toward a pointof intersection. This set of embodiments can be used, for example, tocontrollably mix the fluids as they flow together. The timing ofdelivery and the volume of fluid delivered can be controlled, forexample, by the timing of the vent valve actuation.

Advantageously, the vent valves described herein can be operated withoutconstricting the cross-section of the microfluidic channel on which theyoperate, as might occur with certain valves in the prior art. Such amode of operation can be effective in preventing leaking across thevalve. Moreover, because vent valves can be used, some systems andmethods described herein do not require the use of certain internalvalves, which can be problematic due to, for example, their highexpense, complexity in fabrication, fragility, limited compatibilitywith mixed gas and liquid systems, and/or unreliability in microfluidicsystems. By using an external valve such as a vent valve, macro-scale(rather than micro-scale) mechanical features are employed, which aregenerally less expensive to fabricate and more robust in operation. Inaddition, the external valves described herein function well withheterogeneous fluids (e.g., gas/liquid combinations) and fluidscontaining bubbles, droplets, and/or particles.

In certain embodiments, the fluids used in the systems described hereincan be stored within the systems themselves. While external valves maycontrol the timing of reagent delivery, injection of liquid reagents isnot required to operate some such systems. The ability to operate thesystems without making external connections to fluid sources can greatlysimplify operation.

The articles and systems described herein may be produced inexpensivelyand, in some cases, may be disposable. In addition, the articles andsystems described herein can be fabricated quickly due to the absence ofcomplex mechanical features, in some embodiments. These advantages canallow one to test and implement a wide range of configurations, whichmay be suitable for a large number of chemical and biological systems(e.g., biological assays). Other advantages are described in more detailbelow.

The systems and methods described herein may find application in avariety of fields. In some cases, the systems and methods can be used tocontrol fluid flow and mixing in a variety of microfluidic systems suchas, for example, microfluidic point-of-care diagnostic platforms,microfluidic laboratory chemical analysis systems, fluidic controlsystems in cell cultures or bio-reactors, among others. The articles,systems, and methods described herein may be particularly useful, insome cases, wherein an inexpensive, robust, disposable microfluidicdevice is desired. The fluid control described herein may be used toperform any suitable chemical and/or biological reaction. As a specificexample, the fluid control described herein may be used to controlreagent transport in antibody assays that employ unstable reactionprecursors, such as the silver solution assay described in the Examplessection.

The articles, components, systems, and methods described herein may becombined with those described in International Patent Publication No.WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod”; International Patent Publication No. WO2005/072858(International Patent Application Serial No. PCT/US2005/003514), filedJan. 26, 2005 and entitled “Fluid Delivery System and Method”;International Patent Publication No. WO2006/113727 (International PatentApplication Serial No. PCT/US06/14583), filed Apr. 19, 2006 and entitled“Fluidic Structures Including Meandering and Wide Channels”; U.S. patentapplication Ser. No. 12/113,503, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems” and published as U.S. PublicationNo. 2008/0273918; U.S. patent application Ser. No. 12/196,392, filedAug. 22, 2008, entitled “Liquid containment for integrated assays” andpublished as U.S. Publication No. 2009/0075390; U.S. patent applicationSer. No. 12/428,372, filed Apr. 22, 2009, entitled “Flow Control inMicrofluidic Systems” and published as U.S. Publication No.2009/0266421; U.S. patent application Ser. No. 12/640,420, filed Dec.17, 2009, entitled, “Reagent Storage in Microfluidic Systems and RelatedArticles and Methods” and published as U.S. Publication No.2010/0158756; and U.S. patent application Ser. No. 12/698,451, filedFeb. 2, 2010, entitled, “Structures for Controlling Light Interactionwith Microfluidic Devices” and published as U.S. Publication No.2010/0196207; U.S. Patent Apl. Ser. No. 61/325,023, filed Apr. 16, 2010,entitled, “Feedback Control in Microfluidic Systems”; U.S. Patent Apl.Ser. No. 61/325,044, filed Apr. 16, 2010, entitled, “System for Analysisof Samples”; and U.S. Patent Apl. Ser. No. 60/363,002, filed Jul. 9,2010, entitled, “Systems and Devices for Analysis of Samples”, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

A series of exemplary devices including vent valves and other componentsare now described.

FIG. 1 includes an exemplary schematic illustration of a devicecomprising one or more vent valves and one or more fluids, according toone set of embodiments. In the set of embodiments illustrated in FIG. 1,a device 10 includes a channel 12 comprising an inlet 14, an outlet 15,an upstream portion 16, and a downstream portion 18. The channel canalso contain a fluid in at least one of the upstream and downstreamchannel portions, such as a first fluid 20. The channel can alsocontain, in addition to or in place of the first fluid, a second fluid22. In embodiments in which multiple fluids are stored, the fluids canbe separated from one another by one or more immiscible separating fluidplugs (e.g., a separation fluid such as a gas (e.g., air) or an oil). Insome instances, the device (including any inlets, outlets, and ventvalves) is sealed and is constructed and arranged for storing a fluid(e.g., either or both of fluids 20 and 22) in the device prior to firstuse of the device by an intended user.

As shown illustratively in FIG. 1, first fluid 20 and second fluid 22are not in direct contact with each other. For example, the first andsecond fluids within the channel may be separated by a third fluid 21that is immiscible with both the first and second fluids. In one set ofembodiments, fluids 20 and 22 can both be liquids separated by, forexample, a plug of gas positioned between them. In another embodiment,fluids 20 and 22 are liquids separated by a third liquid that isimmiscible with both liquids. When more than two fluids are employed,any suitable combination of gases and liquids can be used to separatemultiple portions of fluid within the channel(s).

Device 10 also comprises a vent valve 24 positioned between thedownstream and upstream channel portions. As used herein, a “vent valve”refers to a valve that comprises a port in fluid communication with achannel, and a mechanism that can be operated to open and close theport, wherein the vent valve exposes the channel interior to, or sealsthe channel interior from, an environment external to the channelinterior. Exemplary exterior environments can include, for example, anambient environment (e.g., air) and a reservoir containing a fluid(e.g., a pressurized or unpressurized gas).

FIGS. 2A-2F include exemplary cross-sectional schematic illustrations ofa vent valve. In the set of embodiments illustrated in FIGS. 2A-2B, ventvalve 24A is positioned adjacent channel 12. The vent valve includes aport 26A in fluid communication with the channel. In addition, the ventvalve includes a seal 28A (e.g., a cover) which can be moved by anactuator 30A. In FIG. 2A, the vent valve is open such that channel 12 isexposed to an ambient environment 32 via port 26A. In FIG. 2B, the ventvalve is closed such that channel 12 is isolated from ambientenvironment 32 by seal 28A. As shown in the illustrative embodiments ofFIGS. 2C-2D, vent valve 24B includes a seal 28B in the form of a plugthat can block an opening of port 26B. Seal 28B may be deformable insome embodiments.

As shown in the illustrative embodiments of FIGS. 2E-2F, vent valve 24Cincludes a valving mechanism 31 operatively associated with a tube 33defining a channel (e.g., a microfluidic channel) permitting fluid flow.The tube is attached to a plate 35 which, when pressed against themicrofluidic substrate (e.g., exterior surface 27), may form afluid-tight seal. The seal may be formed using a compressed gasket oro-ring 37, or any other suitable component as described in more detailbelow. Alternatively, the tube may be press-fit into the port. As shownin FIGS. 2E-2F, the valve is in fluidic communication with port 26C. Thevalve can be opened or closed by actuating valving mechanism 31. Whenthe valve is open, e.g., as illustrated in FIG. 2E, fluid in tube 33 canfreely flow across the valving mechanism. In such and other embodiments,channel 12 is exposed to and in fluid communication with an environment39 at the other end of the tube. When the valve is closed, e.g., asillustrated in FIG. 2F, fluid in tube 33 can no longer flow across thevalving mechanism; therefore, channel 12 is isolated from and no longerin fluid communication with environment 39 at the other end of the tube.It should be appreciated that environment 39 may be any suitableenvironment, including an ambient environment (e.g., the tube can beopen to air) and a reservoir containing a fluid (e.g., a gas such ascompressed air or nitrogen).

One of ordinary skill in the art would be capable of selecting asuitable actuation mechanism and/or seal to be used for a specificapplication. Non-limiting examples of a valving mechanism which may beoperatively associated with a tube or other suitable component of a ventvalve include a diaphragm valve, ball valve, gate valve, butterflyvalve, globe valve, needle valve, pinch valve, poppet valve, or pinchvalve. The valving mechanism may be actuated by any suitable means,including a solenoid, a motor, by hand, or by hydraulic/pneumaticpressure. Additionally, any suitable seal may be used. In someembodiments, the seal can comprise a rubber or other elastomericmaterial which can be, in some cases, selected to be compatible with oneor more fluids within the system. Suitable seal materials include, butare not limited to, natural rubbers, thermoplastics, synthetic rubbers(e.g., fluoropolymers, neoprene, nitrile, silicone, fluorosilicone,etc.), or combinations of these. The seal can be affixed to orintegrally formed on a surface of the vent valve, in some embodiments.In some cases, the seal can comprise a lip (not shown) on a surface ofthe vent valve designed to engage a corresponding notch on a surface ofthe device (or vice versa) such that when the vent valve is in a closedposition, the lip engages the notch to form a seal.

In some cases, one or more vent valves can be electronically actuated.For example, in some embodiments, a sensor may be in operativeassociation with an actuator and/or a microprocessor able to open orclose the vent valve in response to a signal determined within thesystem. In some cases, a vent valve may be electronically actuated basedupon timing dictated, for example, by a pre-determined program executedby a microprocessor. It is to be understood that any suitable controlsystem and technique disclosed herein can potentially be provided incombination with other control systems not specifically described toprovide other or additional functionality.

The vent valve can be positioned, in some cases, such that the port islocated adjacent (e.g., over) at least a portion of the microfluidicchannel. For example, in some embodiments, the port can comprise anopening connecting the channel interior to an exterior surface 27 of thedevice in which the channel is formed, as illustrated in FIGS. 2A-2B.Although FIGS. 2A-2B show an opening of the port being directly adjacentexterior surface 27, in other embodiments such as those shown in FIGS.2C-2D, an opening of a port can be connected to a channel interior by anintervening channel 29. In some embodiments, a channel is formed in anarticle, and the port can be formed such that it extends in a directionthat is substantially out of the plane of the article. For example, insome embodiments, the port may be formed by drilling a hole into the topsurface of a substrate in which the channel is formed. In otherembodiments, the port can be molded into a substrate fabricated byinjection molding using a pin located in the mold cavity, e.g., asdescribed in Example 1.

The vent valve can be used to control the movement of fluid within achannel system. Referring back to FIG. 1, a vacuum can be applied tooutlet 92 (with outlet 15 closed, or to 15 with outlet 92 closed), whichcan pull fluid 22 toward the outlet in the direction of arrow 52. Whenvent valve 24 is open, a fluid from an environment exterior to thechannel interior can be drawn through the vent valve, and into thechannel. For example, when the fluid in the exterior environment isambient air, the air can enter into the channel interior upon opening ofthe vent valve. In some cases, this fluid from the external environmentcan mix with a fluid inside the channel system. For instance, inembodiments in which fluid 21, which is positioned at vent valve 24, isa gas, the ambient air entering into the channel can mix with fluid 21.

In some cases, such as when the port of the vent valve is in fluidcommunication with ambient air, the resistance to the flow of fluid 21or any other fluid adjacent to fluid 20 may be smaller than theresistance to flow of fluid 20 itself, and in such cases fluid 20 canremain substantially stationary inside the channel even when a source ofvacuum is applied downstream of fluid 20. This can allow for the flow offluid 22 through the downstream portion of the channel withoutsubstantially flowing fluid 20. When vent valve 24 is closed, ambientair can no longer be drawn into the channel through the vent valve, andfluid 20 is transported through channel 12 in the direction of arrow 52.

In some embodiments, a device described herein includes a plurality ofvent valves. A device may comprise, for example, multiple vent valvespositioned in series along a main channel between an inlet and an outletof the main channel. The set of embodiments illustrated in FIG. 1, forexample, includes an optional second vent valve 34 that is positioned inseries with vent valve 24, between inlet 14 and outlet 15, along channel12.

In some cases, a device can include one or more branching channels,i.e., channels that intersect with another channel of the device at apoint of intersection. For example, in some embodiments, the devicecomprises a first upstream portion comprising a first branching channeland a second upstream portion comprising a second branching channel. Thefirst and second branching channels can intersect with each other, insome cases. In addition, one or more branching channels can befluidically connected with a downstream channel portion. In some cases,a device includes one or more branching channels in fluid communicationwith a main channel, any of which can contain a one or more fluidsstored therein (e.g., prior to first use). For example, in the set ofembodiments illustrated in FIG. 1, device 10 optionally includeschannels 36 and/or 38, which branch from main channel 12. Channels 36and 38 intersect at the location of optional vent valve 34, and arefluidically connected to the downstream portions of channel 12 (e.g.,downstream portion 18). Each of the branching channels can also includebranching channels, in some embodiments. For example, any of channels40, 42, and 44, which branch from channel 36 may be included in thedevice. In addition, any of channels 46, 48, and 50, which branch fromchannel 38 may be included in the device in some instances. Optionally,one or more vent valves may be associated with the one or more branchingchannels. Additional layouts of vent valves and channels, as well asfunctionalities associated with the same, are described in more detailbelow.

In one set of embodiments, an upstream channel portion (e.g., of a mainchannel) can serve as a first branching channel, and the device canfurther comprise a second branching channel, wherein the first andsecond branching channels connect at an intersection and are fluidicallyconnected to a downstream channel portion. In the set of embodimentsillustrated in FIG. 1, upstream portion 16 of main channel 12 can serveas the first branching channel, while either or both of channels 36 and38 can serve as second (or third) branching channels.

The channel layouts described herein can be used to store fluids in anysuitable configuration. Any of the branching channels can contain one ormore fluids in place of or in addition to one or more fluids which maybe contained within the main channel. For example, a first fluid can becontained in a main channel, and a second fluid can be contained withina first branching channel. In some cases, a third fluid can be containedin a second branching channel, and so on. For example, in the set ofembodiments illustrated in FIG. 1, upstream portion 16 can containoptional fluid 60, optional branching channel 36 can contain optionalfluid 62, and optional branching channel 38 can contain optional fluid64. In addition, optional branching channels 40, 42, and 44 can containoptional fluids, 66, 68, and 70, respectively, and optional branchingchannels 40, 42, and 44 can contain optional fluids, 72, 74, and 76,respectively. In some cases, one or more of such fluids can be storedand sealed in the device prior to first use.

Vent valves can be positioned in any suitable location within a device.In some cases, vent valves are positioned between two fluids (e.g., twostored fluids). For example, in the set of embodiments illustrated inFIG. 1, vent valve 24 is positioned between first fluid 20 and secondfluid 22. Additionally or alternatively, optional vent valve 34 can bepositioned between optional third fluid 60, and first fluid 20 and/orsecond fluid 22. In some cases, a vent valve is positioned between aportion of a first branching channel and a portion of a main channel.For instance, a vent valve can be positioned at the intersection of twoor more channels, such as at the intersection of a branching channel anda main channel. For example, in FIG. 1, optional vent valve 34 ispositioned at the intersection of channel 12 and optional channels 36and 38. In addition, optional vent valve 78 is positioned at theintersection of optional channels 40, 42, 44, and 36. In some cases, oneor more vent valves can be positioned at a portion of a branchingchannel. For example, in FIG. 1, branching channels 46, 48, and 50include vent valves 80, 82, and 84, respectively, which are positionedat non-intersecting portions of the branching channels.

Methods of transporting and/or mixing fluids are also provided. In oneset of embodiments, a method comprises causing one or more fluids tomove while keeping one or more other fluids substantially stationary.For example, in the set of embodiments illustrated in FIG. 1, a pressuregradient can be applied to channel 12, e.g., by applying a negativepressure to an outlet (e.g., outlet 15 with outlet 92 closed or outlet92 with outlet 15 closed). When vent valve 24 is in the open position,the pressure gradient can cause fluid 22 to flow through channel 12 inthe direction of arrow 52. This can occur without substantially flowingfluid 20 as described herein. In some embodiments, ambient air, having alower resistance to fluid flow than fluid 20 within channel 12, can bepulled through vent valve 24, allowing fluid 20 to remain substantiallystationary. In some embodiments, a second fluid from a portion of thechannel upstream of the portion from which the first fluid is flowed canbe transported by actuating a vent valve between the upstream anddownstream channel portions such that the vent is closed. For example,in FIG. 1, when vent valve 24 is in the closed position and an upstreaminlet (e.g., inlet 14) or vent valve (e.g., vent valve 34) is open, thepressure gradient can cause fluid 20 to flow through channel 12 in thedirection of arrow 52.

The timing of fluid flow can also be controlled using the systems andmethods described herein. For instance, in some embodiments, fluids 22and 20 can be transported through channel 12 substantiallysimultaneously (e.g., by applying a vacuum after closing vent valve 24).In other embodiments, fluids 22 and 20 can be transported throughchannel 12 sequentially (e.g., by first applying a vacuum before closingvent valve 24, thereby transporting fluid 22, and then closing ventvalve 24 to transport fluid 20). These methods can be generally used tocontrol the flow of any fluid within any channel by closing appropriatevent valves between the negative pressure source and the fluid onewishes to flow within the channel. For example, if transport of optionalfluid 62 is desired, a negative pressure can be applied to outlet 92while outlet 15 and vent valves 24 34, and 94 are closed (and while avalve upstream of fluid 62, such as vent valve 78 remains open). In somecases, this transport takes place when other branches such as branches16 and 38 include inlets or vent valves, positioned upstream of anyfluid contained in the branches, are in the closed position, or indevices that do not include other branches such as branches 16 and 38.Using these and other methods, fluids can be transported to a desiredlocation (e.g., a reaction site) within a fluidic system at specific andpredetermined points in time, and in a particular order, to carry out areaction or other fluidic process. Furthermore, the articles and methodsdescribed herein can allow a first set of processes to be decoupled froma second set of processes. For instance, the time of mixing of two ormore fluids within one or more mixing regions can be decoupled from thetime of incubation of a sample within a reaction area, as each of theseprocesses can be controlled independently. Further advantages andexamples are provided herein.

Methods of mixing two or more fluids are also provided. Mixing mayinvolve the use of branching channels in some cases. In someembodiments, a method comprises providing a device with a main channel,a first branching channel containing a first fluid, and a secondbranching channel containing a second fluid, wherein the first andsecond branching channels connect at an intersection and are fluidicallyconnected to the main channel. In some embodiments, the first branchingchannel can include a portion of a main channel that is upstream of theintersection. For example, in the set of embodiments illustrated in FIG.1, the main channel can comprise channel 12, while the first branchingchannel can comprise upstream portion 16 (containing fluid 60), and thesecond branching channel can comprise channel 36 (containing fluid 62).In some cases, the first and second branching channels both deviate indirection from the main channel. For example, in FIG. 1, the mainchannel can comprise channel 12, with the first branching channelcomprising channel 36 (containing fluid 62), and the second branchingchannel comprising channel 38 (containing fluid 64). In someembodiments, the device can include a vent valve positioned between aportion of the first branching channel and a portion of the mainchannel. In some cases, the vent valve can be positioned at theintersection of the first and second branching channels. For example, inFIG. 1, vent valve 34 is positioned at the intersection of channels 12,38, and 36. In some embodiments, the vent valve can be positionedupstream from an intersection of the branching channels. For example, inFIG. 1, optional vent valve 94 is positioned over channel 36, upstreamof the intersection of channels 36 and 38. In some instances, the devicecan include a vent valve positioned between a portion of the secondbranching channel and a portion of the main channel. In FIG. 1, ventvalve 34 is positioned between second branching channel 38 and mainchannel 12. In addition, optional vent valve 96 is positioned between aportion of second channel 38 and main channel 12.

In some embodiments, a method of mixing can comprise actuating at leastone vent valve while providing a pressure gradient across two openingsin the device (e.g., an inlet and an outlet) to cause first and secondfluids to flow into an intersection of two or more channels. The flow ofthe first and second fluid into the intersection may occur substantiallysimultaneously. In some cases, at least a portion of each of the fluidstransported to the intersection can be mixed to produce a mixed fluid. Asingle vent valve can be actuated to cause the flow of two or morefluids. For example, in FIG. 1, when vent valve 34 is closed (andoptional vent valves 94 and 96 are absent) two or more of fluids 62, 60,and 64 can be flowed toward the intersection of channels 12, 36, and/or38, as long as at least one inlet or vent valve upstream of each ofthese fluids are open. As another example, when optional vent valve 78is closed (assuming other vent valves between valve 78 and the pressuregradient source are also closed) two or more of fluids 66, 68, and 70can be transported to the intersection of channels 40, 42, and/or 44 aslong as at least one inlet or vent valve upstream of each of thesefluids are open.

In some embodiments, a device may include a main channel, a firstbranching channel containing a first fluid, a second branching channelcontaining a second fluid, wherein the first and second branchingchannels connect at an intersection and are fluidically connected to themain channel. A third fluid may optionally be provided in the mainchannel, which may be, for example, downstream of the branchingchannels. A vent valve may be positioned between a portion of the firstbranching channel and a portion of the main channel (e.g., at theintersection of the first and second channels, or along the mainchannel). Operating the system may involve actuating the vent valve,causing the first and second fluids to flow into the intersectionsubstantially simultaneously, and mixing at least portions of the firstand second fluids to produce a mixed fluid. In some embodiments, thethird fluid in the main channel may be flowed before actuating the ventvalve (or a series of vent valves) without substantially flowing thefirst and second fluids. After the third fluid is flowed in the mainchannel (e.g., towards a reaction site or other portion of the device),the vent valve that is positioned between a portion of the firstbranching channel and a portion of the main channel may be actuated toallow the flow of the first and second fluids as described above. Insome instances, a substantially constant vacuum is applied at the outletof the main channel and timing of the flow of the third, second, andfirst fluids is accomplished by timing of actuation of the vent valve.Operating the system may include, in some cases, waiting a predeterminedtime after actuating the vent valve in order to allow for apredetermined amount of mixing (e.g., such that not all of the first andsecond fluids are allowed to combine), and then opening the vent valveto stop the flow of the remaining first and second fluids in the firstand second branching channels, respectively, from flowing into the mainchannel. Accordingly, a predetermined mixed amount of the first andsecond fluids may be delivered to the main channel using this method oftiming.

In some embodiments, multiple vent valves are actuated to cause the flowof two or more fluids toward a channel intersection. For example, inFIG. 1, vent valves 94 and 96 can both be closed (e.g., substantiallysimultaneously), which can cause fluids 62 and 64 to flow toward theintersection of channels 36 and 38 (e.g., substantially simultaneously).Inlet 14, if present, can also remain closed. The fluids may flow due tothe presence of a pressure gradient, which may be formed by, forexample, applying a substantially constant reduced pressure at outlet92, and keeping all other inlets, outlets or vent valves between thefluids and outlet 92 closed. In addition, vent valves 80, 82, and 84 canbe closed (e.g., substantially simultaneously) to cause fluids 72, 74,and 76 to flow toward portion 98 of channel 38 (e.g., substantiallysimultaneously). In certain embodiments, the fluids reach a commonregion (e.g., an intersection, a mixing region, etc.) substantiallysimultaneously. Substantially simultaneous transport and/or delivery oftwo or more fluids to a common region can be useful in achievingefficient mixing of the two fluids, for example, by maximizing thecommon surface area between two or more fluids. In addition,substantially simultaneous delivery of two or more fluids to a commonregion can aid in delivering substantially equivalent volumes of two ormore fluids, as is discussed in more detail below. This can be importantin processes that require the mixing of precise volumes of fluid. Insome cases, substantially simultaneous delivery of two or more fluids toa common region helps to avoid the formation of bubbles between themixed fluid and other fluids within the system, as described in moredetail below.

One or more parameters of a device can be chosen, in some cases, suchthat two or more fluids transported through a device contact each otherwithin a region of the device substantially simultaneously. For example,in some cases, the cross-sectional areas of at least two channels (e.g.,two branching channels, a branching channel and a main channel, etc.),the viscosities of the fluids to be mixed, the relative volumes of thefluids to be mixed, the linear lengths of the channels containing thefluids to be mixed, the amount of pressures applied, and the distancesfrom each of the fluids to the point of intersection are selected suchthat, when equal pressures are applied to each of the two channels, thefluids within them flow into an intersection or other common regionsubstantially simultaneously.

In order to control mixing within the system, it may be useful tocontrol the flow rates of the fluids in the system. Problems can arise,for example, if one fluid (e.g., fluid 62 in FIG. 1) reaches a commonarea such as a vent valve before another fluid (e.g., fluid 60 in FIG.1). In such cases, mixing might not occur as anticipated. For example,in some cases, the first fluid (e.g., fluid 62), upon reaching ventvalve 34 before a second fluid (e.g., fluid 60), can fill the vent valveand effectively trap a bubble of a separating fluid plug between thevent valve and the front end of the second fluid. In this case, aportion of fluid 62 will be separated and flowed down the main channelwithout mixing with fluid 60. In some embodiments, this can lead toexposing the reaction area or other area of analysis to a first volumeof an unmixed reagent (e.g., a reagent in fluid 62), followed by asegment of a separating fluid plug, followed by a substantiallyirreproducible mixture of fluids 60 and 62. In some such cases, theresulting chemical or biological reaction in the reaction area may beirreproducible.

Without wishing to be bound by theory, the inventors believe that thefollowing theory can be used to better understand the relationshipbetween flow rate, channel dimensions, and viscosities of fluids flowingin a channel system. Laminar flow of an incompressible uniform viscousfluid (e.g., Newtonian fluid) in a tube driven by pressure can bedescribed by Poiseuille's Law, which is expressed as follows:

$\begin{matrix}{Q = {\frac{\pi \; R^{4}}{8\eta} \cdot \frac{\Delta \; P}{L}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

where Q is the volumetric flow rate (in m³/s, for example), R is theradius in of the tube (m), ΔP is the change in pressure across the tube(Pa), η is the dynamic fluid viscosity (Pa·s), and L is the length ofthe tube (m). To generalize beyond circular tubes to any closed channel,this equation can be expressed as:

$\begin{matrix}{Q = {\frac{{AR}_{H}^{2}}{8\eta} \cdot \frac{\Delta \; P}{L}}} & \left( {{Equation}\mspace{14mu} 1b} \right)\end{matrix}$

where A is the cross-sectional area of the channel and R_(H) is thehydraulic radius, R_(H)=2A/P with P being the parameter of the channel.For a circular tube, AR_(H) ²=πR⁴. For a rectangular channel of width wand depth d, AR_(H) ²=(wd)³/(w+d)². When performing a controlled mixingof multiple fluids, it is important to consider the factors impactingthe flow of each individual fluid. In a system designed such that theΔP, η, R_(H) ² and L are equal, both fluids should flow in a similarmanner and reproducible mixing of the fluid should be achievable. Whenone or some of these parameters differ for the fluids, the design of thesystem should be such that the differences cancel out.

In some embodiments two or more fluids that are to be mixed havesubstantially equivalent volumes. The two or more fluids may also havesimilar viscosities, and may be positioned in channels having similarchannel cross-sections. In some cases, the volume of one or moreseparating fluid plugs between the front interfaces of the fluids to bemixed and the intersection (e.g., mixing chamber) in which they are tobe mixed can be similar for both reagents. This can help ensure thatwhen the fluids begin moving toward an intersection, the fluids reachthe intersection substantially simultaneously. These and otherparameters may allow the two or more fluids to be delivered to a commonarea substantially simultaneously, thereby resulting in reproduciblemixing.

In some embodiments in which a first fluid has a first volume, and asecond fluid has a second volume different than the first volume, thespeed of the smaller volume fluid may increase relative to the largervolume fluid, due to the relatively smaller resistance to fluid flowexhibited by the relatively smaller volume fluid (the hydrodynamicresistance to flow for liquids scales as 1/L, L being the length of thefluid segment; assuming equal channel dimensions and viscosities, theshorter fluid segment will flow faster than the longer fluid segment).This can lead to a deviation from a desired mixing ratio, since it mayresult in a relatively large amount of the smaller volume fluid beingadded, relative to the larger volume fluid. This behavior can beself-amplifying, because as the smaller volume fluid moves faster itsvolume is disproportionately decreased, leading to a further increase inspeed. To overcome this potential problem, the cross sections of thechannels can be selected, or the viscosities of the fluids to be mixedcan be selected, so that there is equal resistance to fluid flow in thechannels. For instance, to increase the resistance to flow of thesmaller volume fluid, the smaller volume fluid may be positioned in achannel having a smaller cross-section than that containing the largervolume fluid in order to match the overall resistance of the largervolume fluid. Additionally or alternatively, the viscosity of thesmaller volume fluid may be increased to increase its resistance tofluid flow to match the overall resistance of the larger volume fluid.

In some cases, the transport and/or mixing of fluids within a channelcan be enhanced by employing a channel with a relatively small amount ofsurface roughness. Inhomogeneities in the surface of a channel (e.g.,changes in roughness, imperfections in channel surfaces, chemicaldeposits on a channel surface, etc.) between the storage location ofeach of the liquids and the mixing chamber, can affect the advancementof the interfaces between the fluid portions and the separating fluidplug (and thus, the bulk of the liquids). As such, in some embodimentsdescribed herein, a channel surface has a relatively low surfaceroughness. The surface of a channel may have a root-mean-square (RMS)surface roughness of, for example, less than about 5 μm. In otherembodiments, the RMS surface roughness may be less than about 3 μm, lessthan about 1 μm, less than about 0.8 μm, less than about 0.5 μm, lessthan about 0.3 μm, or less than about 0.1 μm.

The addition of wetting agents to a fluid can also promote reproducibleadvancement of a fluid within a channel. The wetting agents canstabilize the interface between the fluid and the separating fluid plugand/or reduce the impact of inhomogeneities on a surface of a channel.In some embodiments, the wetting agent can be selected such that it doesnot adversely react with one or more components (e.g., a reagent) withina fluid. Examples of suitable wetting agents include, but are notlimited to, non-ionic detergents (e.g., poly(ethylene oxide) derivativeslike Tween 20 and Triton, fatty alcohols), anionic detergents (e.g.,sodium dodecyl sulfate and related detergents with shorter or longeralkane chains such as sodium decyl sulfate or sodium octadecyl sulfate,or fatty acid salts), cationic detergents (e.g., quaternary ammoniumcations such as cetyl trimethylammonium bromide), zwitterionicdetergents (e.g., dodecyl betaine) and perfluorodetergents (e.g.,Capstone FS-10).

Additionally or alternatively, the surface of a channel can be treatedwith a substance to facilitate inhibition or enhancement of fluid flow(e.g., hydrophobic or hydrophilic reagents).

In some embodiments, unpredictable fluid behavior can be inhibited byemploying relatively fast flow rates of the fluids within a channel. Theflow rate may depend on factors such as the viscosities of the fluids tobe transported, the volumes of the fluids to be transported, the crosssectional areas and/or cross sectional shapes of the channels containingthe fluids, the pressure gradient, among other factors. In some cases,at least one fluid within a channel is transported at a linear flow rateof at least about 1 mm/s, at least about 5 mm/s, at least about 10 mm/s,or at least about 15 mm/s, at least about 25 mm/s, or at least about 100mm/s. The linear flow rate may, in some embodiments, be between about 1mm/s and about 100 mm/s, between about 5 mm/s and about 100 mm/s,between about 10 mm/s and about 100 mm/s, between about 15 mm/s andabout 100 mm/s, between about 1 mm/s and about 25 mm/s, between about 5mm/s and about 25 mm/s, between about 10 mm/s and about 25 mm/s, orbetween about 15 mm/s and about 25 mm/s. Different flow rates may beimplemented at different points in time depending on the fluid beingtransported and/or the process to be carried out in a device. Forinstance, in one set of embodiments it may be desirable for a sample tobe flowed through a reaction area relatively slowly (e.g., 0.5 mm s⁻¹)during a first step, but for two fluids to mix in a mixing region at arelatively higher flow rate (e.g., 15 mm s⁻¹) during a second step. Thevent valves and other articles and methods described herein may be used,optionally in combination with the systems and methods described in U.S.patent application Ser. No. 12/428,372, filed Apr. 22, 2009, entitled“Flow Control in Microfluidic Systems” and published as U.S. PublicationNo. 2009/0266421, which is incorporated herein by reference, to controland implement such flow rates and change in flow rates during operationof the device. Two linear flow rates applied during two different stepsof a process carried out in a device may have a difference of, forexample, greater than 1×, 5×, 10×, 15×, 20×, 25×, 30×, 40×, or 50×. Forexample, a relatively high linear flow rate of 15 mm s⁻¹ is 30× fasterthan a relatively slow linear flow rate of 0.5 mm s⁻¹. In some cases,such fluid control is achieved using one or more vent valves, optionallyeven when a source of pressure or reduced pressure (e.g., vacuum) isapplied substantially constantly to the device during the one or moresteps.

As described herein, the intersection of two or more channels cancomprise a mixing region. Such a region can be useful in promotingmixing of multiple fluids that are flowed from multiple channels to theintersection. In some embodiments, the mixing region can have a largercross-sectional area than either of the first or second (or third,fourth, etc.) channels (e.g., branching channels) that intersect at themixing region. For example, a mixing region may have an averagecross-sectional area that is at least 1.2 times, at least 1.5 times, atleast 1.7 times, at least 2 times, at least 3 times, or at least 5 timesthe average cross-sectional area of the largest channel intersecting themixing region. A mixing chamber at the intersection comprising arelatively large volume can help, for example, in compensating for amismatch in the arrival times of two or more fluids at an intersectionof two or more channels.

In other embodiments, however, a relatively smaller mixing region may bepresent in devices described herein. For example, a mixing region mayhave an average cross-sectional area that is less than 5 times, lessthan 3 times, less than 2 times, less than 1.7 times, less than 1.5times, or less than 1.2 times the average cross-sectional area of thelargest channel intersecting the mixing region. In some cases the mixingregion has an average cross-sectional area that is substantially thesame as the average cross-sectional area of the largest channelintersecting the mixing region.

In some cases, the mixing region can comprise a vent valve. For example,the port of a vent valve can provide a volume in which multiple fluidsare mixed. In some embodiments, the cross-sectional area, length, orother parameter of a component (e.g., a channel, a vent valve component(e.g., a port), a mixing region, etc.) can be chosen such that a desiredmixing result is achieved upon flowing two or more fluids within thecomponent. For example, in some embodiments, the volume of the ventvalve (e.g., a port of a vent valve, or an intervening channel of thevent valve connecting a main channel to an opening of the vent valve)can be chosen such that complete mixing of two or more fluids can beachieved (e.g., via diffusion) during their residence time within thevent valve. The volume of the vent valve, including any interveningchannels, may be, for example, less than about 50 μL, less than about 20μL, less than about 10 μL, less than about 5 μL, less than about 3 μL,less than about 1 μL, less than about 0.1 μL, less than about 0.01 μL,less than about 10 nL, or less than about 1 nL. Other volumes are alsopossible.

In a laminar flow environment (which is common to most microfluidicsystems), the mixing of reagents relies mostly on diffusion. In thiscontext, mixing between reagents gradually increases as the reagentsflow together along a channel. In such cases, the length of a mainchannel (e.g., between the vent where mixing occurs and the point of useof the mixed reagents, such as the reaction area) can be chosen suchthat complete or sufficient mixing of two or more fluids can be achieved(e.g., via diffusion) during their residence time within the channel.

Mixing based on diffusion can also be increased by increasing thecombined fluid's residence time in the channel. In some cases, anincubation step can be added to the system. For example, in a systemwith a substantially constant vacuum applied at outlet 92, and havingcombined two liquids upstream of vent valve 34 (with vent valves 34, 24,and 15 closed), these liquids can be incubated in channel 12 by openingvent valve 15 (or optionally opening vent valve 24). By opening ventvalve 15 (or 24), air would preferentially be drawn toward outlet 92through vent valve 15 (or 24), thereby allowing the liquids to remain inplace in channel 12. After sufficient incubation, vent valve 15 (or 24)could be closed, thereby causing the liquids to flow into reaction area86. Advantageously, as illustrated in such and other embodiments,control of fluid flow can be achieved even when a substantially constantvacuum or other source of fluid flow is applied to the device.

In some embodiments, the flow of one or more fluids into an intersectionor other suitable mixing region can be cut off prior to passing theentire volume of fluid to the intersection or mixing region. This may beaccomplished, for example, by opening a vent valve while portions of thefluid are in the channel on opposites sides of the vent valve. Forinstance, a first portion of the fluid may be located in a first channelportion of an underlying channel and a second portion of the fluid maylocated in a second channel portion of the underlying channel, the firstand second channel portions being on opposite sides of the vent valve.When the vent valve is opened while a fluid is underneath its port, afluid from an environment external to the channel interior, such asambient air, can be transported through the port and into the channelinterior if the resistance to fluid flow of the fluid in the externalenvironment is less than the resistance to fluid flow of the portion ofthe remaining portion of the fluid under the vent valve. For example, byintroducing a segment of gas into the channel, the fluid contained inthe channel can divide into first and second portions which areseparated by the segment of gas.

FIGS. 3A-3D include schematic illustrations of a method by which theflow of a fluid can be cut off by actuating a vent valve. In the set ofembodiments illustrated in FIGS. 3A-3D, channel 100 includes inlet 102,outlet 104, and vent valve 106. In addition, channel 100 contains fluid108. The direction of fluid flow in FIGS. 3A-3D is indicated by thearrows. In FIG. 3A, vent valve 106 is open, causing external fluid toflow into the channel via vent valve 106 when negative pressure isapplied to outlet 104. In FIG. 3B, vent valve 106 is closed while inlet102 is open, causing fluid 108 to flow through channel 100 toward outlet104. In FIG. 3C, vent valve 106 is opened before fluid 108 has passedcompletely past the vent valve, causing external fluid to pass throughthe port of the vent valve and into the channel, separating segment 110from fluid 108. Repeating this process can produce multiple fluidsegments from an original single fluid. For example, in FIG. 3D, fluidsegments 110, 111, 112, and 113 have been produced from fluid 108 byclosing and opening vent valve 106 four times. Such methods can be usedto produce one or more fluid portions with a pre-selected length,volume, or other suitable property.

Generating a series of fluid segments or portions from a single fluidsegment can, in some cases, improve the mixing of two or more componentswithin the fluids compared to that in the single fluid segment. Forexample, it is known that components (e.g., particles, reagents, orother entities) within segments of fluid, as might be observed insegmented flow, experience recirculation within the segment duringlinear flow of the segment. In some embodiments, a fluid containing twoor more components to be mixed can be passed under a vent valve, and thevent valve can be opened and closed so as to produce multiple portionsof the fluid, for example, to enhance the mixing of the two or morecomponents within each fluid portion. This feature can be particularlyadvantageous in systems in which turbulent flow is absent (e.g., in manymicrofluidic systems).

The opening of and closing of a vent valve to create separated fluidportions can be useful outside the context of mixing as well. Multipleplugs of single reagent have been shown to be preferable to a singlelong plug in certain situations such as those described, for example, inInternational Patent Publication No. WO2005/072858 (International PatentApplication Serial No. PCT/US2005/003514), filed Jan. 26, 2005 andentitled “Fluid Delivery System and Method,” which is incorporatedherein by reference in its entirety for all purposes. As a specificexample, multiple portions of a rinsing fluid can provide better rinsingor washing of a surface compared to a single, longer fluid portion insome embodiments.

Separating a single fluid portion into two or more fluid portions can beused, in some cases, to produce a suitable volume of fluid for mixingwithin a mixing or other region. For example, in some cases, a firstbranching channel can comprise a first fluid, and a second branchingchannel can comprise a second fluid with a volume substantially largerthan the first fluid. The first and second fluids can be flowed towardan intersection of the first and/or second branching channel and a mainchannel. In some embodiments, prior to passing the entire volume of thefirst and/or second fluid across the intersection, at least one ventvalve in the first or second branching channel can be opened such thatthe first and/or second fluids are divided into first and secondsegments. In other embodiments, prior to passing the entire volume ofthe first and/or second fluid across the intersection, at least one ventvalve in the second branching channel can be opened such that secondfluid is divided into smaller segments (e.g., to match the volume of thefirst fluid). Only one of the segments of second fluid can be deliveredto the intersection to combine with all or portions of the first fluid.These and other methods can allow, in some cases, equal or otherappropriate volumes of the first and second fluids to be delivered tothe main channel, a mixing region, a reaction area, or any othersuitable destination (e.g., when the first and second fluids aredelivered substantially simultaneously to a common region). Thus, insome embodiments, a portion, but not all, of the first fluid, and/or aportion, but not all, of the second fluid, are combined together to forma mixed fluid that is used or delivered to suitable destination.

One example of a method for delivering substantially equal volumes ofmultiple fluids to a common region (e.g., an intersection of two of morechannels) is illustrated schematically in FIGS. 4A-4B. In FIG. 4A, mainchannel 200 includes outlet 202, and is fluidically connected tobranching channels 204 and 206 at vent valve 208. Branching channel 204comprises inlet 210 and contains fluid 212, while branching channel 206comprises inlet 214 and contains fluid 216. In FIG. 4A, fluid 212 issubstantially smaller in volume than fluid 216. In FIG. 4A, vent valve208 is open, allowing exterior fluid to flow through the vent valve andthrough main channel 200 (as indicated by the arrows) upon applicationof negative pressure to the outlet. In FIG. 4B, vent valve 208 isclosed, while inlets 210 and 214 are open, causing fluids 212 and 216 toflow toward outlet 202 upon application of negative pressure. In thisset of embodiments, the viscosities of the fluids and thecross-sectional dimensions of channels 204 and 206 are chosen such thatfluids 212 and 216 contact each other substantially simultaneously atthe intersection of channels 204 and 206. In FIG. 4C, vent valve 208 isopened before fluids 212 and 216 have completely passed through theintersection of channels 204 and 206, creating a segment 218 of mixedfluid containing substantially equal parts of fluid 212 and fluid 216.

In some embodiments, multiple portions of mixed fluid can be created byopening and closing vent valve 208 any number of times. Such embodimentsmay be useful, for example, when fluids 212 and 216 do not initiallycontact each other simultaneously at the intersection of the branchedchannels. In some such cases, the first portion of mixed fluid cancomprise more of the first than the second fluid, while subsequentportions of mixed fluid can contain substantially equal amounts of thefirst and second fluids. In some instances, the first portion of mixedfluid is not useful for a downstream process, so it can be diverted awayfrom the main channel or other region of the device. For instance, anunwanted first portion of mixed fluid may be lead towards a branchingchannel that leads to a waste containment region. Fluid flow can beoptionally controlled by the use of one or more valves (e.g., anexternal valve) in combination with methods described herein. One ormore subsequent portions of mixed fluid, which may be useful for adownstream process, can then be delivered to the main channel or otherregion of the device such as reaction area.

One method for diverting a portion of a mixed fluid (or any other fluid)is shown in FIGS. 4D-4I. As shown in the embodiments illustrated inFIGS. 4D-4I, a branching channel 215 having an outlet 220 is included.This outlet may be operatively associated with the same vacuum sourceoperatively associated with outlet 202. For instance, tubing (not shown)may connect each of the outlets to the vacuum source. In some cases, avalving mechanism (not shown) is operatively associated with the tubing.Each outlet is equipped with an individually controlled valve. Tocombine fluids 212 and 216 to form a mixed fluid, the system is operatedwith outlet 202 open and outlet 220 closed (FIG. 4D). Vent valve 208 isclosed (FIG. 4E) to begin mixing and then opened to deliver only a firstportion of fluid 218 into main channel 220 (FIG. 4F). Once the mixedportion is in the main channel, a valving mechanism (not shown)operatively associated with the outlets is actuated to cease fluidcommunication between the vacuum and outlet 202, while allowing fluidcommunication between the vacuum and outlet 220 (FIG. 4G). Since thevacuum is now operating at outlet 220, fluid 218 can be diverted fromthe main channel into branching channel 215 (FIG. 4H). The valvingmechanism operatively associated with the outlets can then be actuatedto allow fluid communication between the vacuum and outlet 202, whileceasing fluid communication between the vacuum and outlet 220 (FIG. 4I).

Separating a single fluid portion into two or more fluid portions canprovide other advantages apart from mixing fluids and producing fluidsegments. For example, in some cases, when the trailing edge of a fluidreaches a vent valve, a slight burst of liquid can be ejected toward thevent valve (e.g., toward a port in the vent valve, toward an actuatorassociated with the vent valve, etc.). In some cases, the ejected liquidcan interfere with the external valving mechanism. While, in some cases,this does not have an immediate effect on the function of the ventvalve, it can, over time, lead to degradation in performance, such as,for example, contamination of the vent valve with a component (e.g., achemical) of the fluid. Upon repeated use of the mechanism (e.g., toperform multiple experiments), such contamination can alter the normalfunction of the external valving mechanism. The inventors havediscovered within the context of the invention that, in someembodiments, by opening the vent valve before all of the fluid haspassed through the channel underneath the valve (e.g., so as to formmultiple fluid segments), little or no trailing edges reach the ventvalve, and no liquid ejection occurs.

The systems, devices, and methods described herein can be used, in someembodiments, to perform one or more chemical and/or biologicalreactions. The devices described herein can comprise additionalcomponents that may be useful for such and other purposes (e.g., bloodsample analysis). In some cases, the device can comprise a reaction areawhich can be, for example, located downstream of a main channel. The setof embodiments illustrated in FIG. 1 includes optional reaction area 86downstream of main channel 12. The reaction area can be fluidicallyconnected to the outlet of the main channel (e.g., outlet 15 in FIG. 1).The reaction area can serve, for example, as a volume in which achemical and/or biological reaction can take place. In some embodiments,a reagent and/or catalyst can be disposed within the reaction area(e.g., immobilized on a wall of the reaction area). For example, in someembodiments, a binding partner can be disposed in a reaction area (e.g.,on a surface, or on or within an entity contained in the reaction area).Exemplary reaction areas that can be used in devices described hereinare provided in International Patent Publication No. WO2006/113727(International Patent Application Serial No. PCT/US06/14583), filed Apr.19, 2006 and entitled “Fluidic Structures Including Meandering and WideChannels” and U.S. patent application Ser. No. 12/113,503, filed May 1,2008 and entitled “Fluidic Connectors and Microfluidic Systems” andpublished as U.S. Publication No. 2008/0273918; U.S. patent applicationSer. No. 12/196,392, filed Aug. 22, 2008, entitled “Liquid containmentfor integrated assays” and published as U.S. Publication No.2009/0075390, which are incorporated herein by reference.

In addition, in some embodiments, a fluid waste chamber can be included,for example, downstream of the reaction area. The fluid waste chambercan be useful, for example, in providing a volume in which used fluidscan be contained such that they do not flow into a negative pressuresource (e.g., a vacuum) during operation of the device. For example, theset of embodiments illustrated in FIG. 1 includes waste chamber 88 thatretains fluids as they are flowed from reaction area 86. Exemplary wastecontainment regions that can be used in devices described herein areprovided in U.S. patent application Ser. No. 12/196,392, filed Aug. 22,2008, entitled “Liquid containment for integrated assays” and publishedas U.S. Publication No. 2009/0075390, which is incorporated herein byreference.

In the set of embodiments illustrated in FIG. 1, a negative pressuresource can be applied, for example, at any of outlet 15, point 90, andoutlet 92. For example, in some cases, fluid 22 in FIG. 1 may contain asample (e.g., a blood sample). The sample can be introduced into thedevice using a variety of methods. Exemplary methods and articles forsample introduction that can be used with devices described herein areprovided U.S. patent application Ser. No. 12/113,503, filed May 1, 2008and entitled “Fluidic Connectors and Microfluidic Systems” and publishedas U.S. Publication No. 2008/0273918; U.S. patent application Ser. No.12/196,392, filed Aug. 22, 2008, entitled “Liquid Containment forIntegrated Assays” and published as U.S. Publication No. 2009/0075390,which are incorporated herein by reference. The sample can first flowinto reaction area 86, and then into waste containment region 88. Thereaction area may have associated with it a detector that is capable ofdetermining a property of a component in the reaction area. The passingof the sample through the reaction area can allow, in some cases,interaction (e.g., binding) between one or more components of the sample(e.g., an antigen) and one or more components in the reaction area(e.g., an antibody). In some embodiments, the component(s) of thereaction area may be in the form of dried reagents stored in thereaction area prior to first use. This interaction may form a productsuch as a binding pair complex. In some cases, this interaction alonecauses a signal to be determined (e.g., measured) by a detectorassociated with the microfluidic system. In other cases, in order for anaccurate signal to be determined by the detector, the product is treatedby one or more reagents. For example, fluid may contain alabeled-antibody that interacts with an antigen of the sample. Thisinteraction can allow the product to be labeled or the signal from theproduct to be amplified.

In some embodiments, the sample and/or reagent(s) are incubated withinthe reaction area for an amount of time. When heterogeneous affinityreactions are employed, for example, the species in the sample will bindto a capture probe immobilized on the surface of the reaction area.Sufficient incubation time can be achieved by, for example, controllingthe time required for the sample to flow through the reaction area. Theflow rate of the system from the vent valve to the vacuum source can bedependent upon the flow rate of the highest relative viscosity fluidthrough the smallest cross sectional area of channel in the system(e.g., acting as a flow bottleneck). In some embodiments, one or moreproperties of the system can be selected such that a desired residencetime of a fluid (e.g., a sample) within the reaction area is achieved.Examples of parameters that can be adjusted to achieve residence timecontrol include, but are not limited to, the volume of sample itself,which can be determined by the availability of sample (e.g., the volumeof a drop of blood for an assay using a fingerpick of blood), ordetermined for convenience for the user; the viscosity of the sample;the pressure difference (Δp) applied to the outlet of the system (forapplication of negative pressure) or applied to the inlet of the system(for application of positive pressure); and the change in the geometry(e.g., cross-sectional area, length, etc.) and location of the flow ratebottleneck. In some embodiments, the system parameters are chosen suchthat the time of mixing of two or more fluids within one or more mixingregions (e.g., a vent valve) of the system is independent from the timeof incubation of the sample within the reaction area.

In some cases, system parameters can be selected such that two or morefluids can be contacted with the reaction area within a predeterminedperiod of time after mixing the two or more fluids. For example, in someembodiments, the mixed fluid can be contacted with the reaction areawithin 10 minutes of mixing the two or more fluids within the mixedfluid. Such embodiments can be useful, for example, when one or morecomponents within the mixed fluid decompose and/or lose theireffectiveness after a relatively short period of time. As a specificexample, in some embodiments a solution of silver salts can be mixedwith a reducing agent to produce an activated silver solution that canbe effectively used within 10 minutes of mixing. A wide variety ofreducing agents have been developed by the photographic industry and canbe used in embodiments described herein. Some of the most commonly usedreducing agents include: hydroquinone, chlorohydroquinone, pyrogallol,metol, 4-aminophenol and phenidone.

As can be seen, it is useful to have mixing conditions and timingindependent of sample incubation times (so that longer incubation doesnot lead to longer mixing times). The advantages of a vent valves andmethods described herein become apparent. In some cases, certaincomponents of a fluidic system such as the dimensions of channels of thereaction area, applied pressure to induce fluid flow, etc. can bedesigned for whatever sample incubation time is necessary in a reactionarea, and timing of mixing of reagents is controlled by one or more ventvalves.

It should be appreciated that a variety of fluids can be used (e.g.,disposed, flowed, stored) in association with devices described herein.In some embodiments, one or more fluids can comprise a sample to beanalyzed. For example, in some cases, a fluid can comprise whole blood.In some cases, a fluid can comprise a reagent (e.g., an antibody fluid),a rinse fluid, or any other suitable fluid. In some cases, a fluid cancomprise a metal solution. For example, a fluid may comprise asuspension of metal particles (e.g., silver, gold, and the like) whichcan form a colloidal suspension. In some cases, a fluid can comprise areducing agent such as, for example, hydroquinone. In some embodiments,one or more of the fluids can be part of a chemical or biological assay.

Each of the fluids within a channel can have substantially similar ordifferent chemical properties. For example, in some embodiments, a firstfluid in the channel can comprise a sample to be analyzed (e.g., blood)while the second fluid comprises a rinsing solution that can be used,for example, to prepare the downstream portion for the passage of athird fluid. In some embodiments, the first fluid contains a firstreagent for a chemical and/or biological reaction, and the second fluidcontains a second reagent for the chemical and/or biological reactionthat is different from the first reagent.

In addition, each of the fluids within the channel can havesubstantially similar or different physical properties. For example, insome embodiments, first and second fluids within the channel havesubstantially different viscosities. Differences in viscosities cancause differences in flow rate upon application of pressure to thechannel.

As noted herein, in some embodiments, microfluidic systems describedherein contain stored reagents prior to first use of the device and/orprior to introduction of a sample into the device. The use of storedreagents can simplify use of the microfluidic system by a user, sincethis minimizes the number of steps the user has to perform in order tooperate the device. This simplicity can allow microfluidic systemsdescribed herein to be used by untrained users, such as those inpoint-of-care settings. Stored reagents in microfluidic devices areparticularly useful for devices designed to perform immunoassays.

As used herein, “prior to first use of the device” means a time or timesbefore the device is first used by an intended user after commercialsale. First use may include any step(s) requiring manipulation of thedevice by a user. For example, first use may involve one or more stepssuch as puncturing a sealed inlet to introduce a reagent into thedevice, connecting two or more channels to cause fluid communicationbetween the channels, preparation of the device (e.g., loading ofreagents into the device) before analysis of a sample, loading of asample onto the device, preparation of a sample in a region of thedevice, performing a reaction with a sample, detection of a sample, etc.First use, in this context, does not include manufacture or otherpreparatory or quality control steps taken by the manufacturer of thedevice. Those of ordinary skill in the art are well aware of the meaningof first use in this context, and will be able easily to determinewhether a device of the invention has or has not experienced first use.In one set of embodiments, devices of the invention are disposable afterfirst use, and it is particularly evident when such devices are firstused, because it is typically impractical to use the devices at allafter first use.

Reagents may be stored and/or disposed in a device in fluid and/or dryform, and the method of storage/disposal may depend on the particularapplication. Reagents can be stored and/or disposed, for example, as aliquid, a gas, a gel, a plurality of particles, or a film. The reagentsmay be positioned in any suitable portion of a device, including, butnot limited to, in a channel, reservoir, on a surface, and in or on amembrane, which may optionally be part of a reagent storage area. Areagent may be associated with a microfluidic system (or components of asystem) in any suitable manner. For example, reagents may be crosslinked(e.g., covalently or ionically), absorbed, or adsorbed (physisorbed)onto a surface within the microfluidic system. In one particularembodiment, all or a portion of a channel (such as a fluid path of afluid connector or a channel of the device substrate) is coated with ananti-coagulant (e.g., heparin). In some cases, a liquid is containedwithin a channel or reservoir of a device prior to first use and/orprior to introduction of a sample into the device.

In some embodiments, dry reagents are stored in one section of amicrofluidic device and wet reagents are stored in a second section of amicrofluidic device. Alternatively, two separate sections of a devicemay both contain dry reagents and/or wet reagents. The first and secondsections may be in fluid communication with one another prior to firstuse, and/or prior to introduction of a sample into the device, in someinstances. In other cases, the sections are not in fluid communicationwith one another prior to first use and/or prior to introduction of asample into the device. During first use, a stored reagent may pass fromone section to another section of the device. For instance, a reagentstored in the form of a fluid can pass from a first section to a secondsection of the device after the first and second sections are connectedvia a fluid path (e.g., a fluidic connector, as described in more detailin U.S. patent application Ser. No. 12/113,503, filed May 1, 2008 andentitled “Fluidic Connectors and Microfluidic Systems” and published asU.S. Publication No. 2008/0273918; U.S. patent application Ser. No.12/196,392, filed Aug. 22, 2008, entitled “Liquid Containment forIntegrated Assays” and published as U.S. Publication No. 2009/0075390,which are incorporated herein by reference). In other cases, a reagentstored as a dried substance is hydrated with a fluid, and then passesfrom the first section to the second section upon connection of thesections. In yet other cases, a reagent stored as a dried substance ishydrated with fluid, but does not pass from one section to anothersection upon connection of the sections.

By maintaining an immiscible fluid (a separation fluid) between each ofthe reagents in the reagent storage area, the stored fluids can bedelivered in sequence from the reagent storage area while avoidingcontact between any of the stored fluids. Any immiscible fluid thatseparates the stored reagents may be applied to the reaction areawithout altering the conditions of the reaction area. For instance, ifantibody-antigen binding has occurred at one of the detection zones ofthe reaction area, air can be applied to the site with minimal or noeffect on any binding that has occurred.

As described herein, storing reagents in a microfluidic system can allowthe reagents to be dispensed in a particular order for a downstreamprocess (e.g., amplifying a signal in a reaction area). In cases where aparticular time of exposure to a reagent is desired, the amount of eachfluid in the microfluidic system may be proportional to the amount oftime the reagent is exposed to a downstream reaction area. For example,if the desired exposure time for a first reagent is twice the desiredexposure time for a second reagent, the volume of the first reagent in achannel may be twice the volume of the second reagent in the channel. Ifa substantially constant pressure differential or source of fluid flowis applied in flowing the reagents from the channel to the reactionarea, and if the viscosity of the fluids is the same or similar, theexposure time of each fluid at a specific point, such as a reactionarea, may be proportional to the relative volume of the fluid. Factorssuch as channel geometry, pressure or viscosity can also be altered tochange flow rates of specific fluids from the channel. The stored fluidscan also be manipulated after storage (e.g., at first use) by a userusing the vent valves and other articles and methods described herein.

Additionally, this strategy of storing reagents in sequence, especiallyamplification reagents, can be adapted to a wide range of chemistries.For example, various amplification chemistries that produce opticalsignals (e.g., absorbance, fluorescence, glow or flashchemiluminescence, electrochemiluminescence), electrical signals (e.g.,resistance, conductivity or impedance of metal structures created by anelectroless process) or magnetic signals (e.g., magnetic beads) can beused to allow detection of a signal by a detector.

Reagents can be stored in a microfluidic system for various amounts oftime. For example, a reagent may be stored for longer than 1 hour,longer than 6 hours, longer than 12 hours, longer than 1 day, longerthan 1 week, longer than 1 month, longer than 3 months, longer than 6months, longer than 1 year, or longer than 2 years. Optionally, themicrofluidic system may be treated in a suitable manner in order toprolong storage. For instance, microfluidic systems having storedreagents contained therein may be vacuum sealed, stored in a darkenvironment, and/or stored at low temperatures (e.g., refrigerated at2-8 degree C., or below 0 degrees C.). The length of storage depends onone or more factors such as the particular reagents used, the form ofthe stored reagents (e.g., wet or dry), the dimensions and materialsused to form the substrate and cover layer(s), the method of adheringthe substrate and cover layer(s), and how the device is treated orstored as a whole.

In some embodiments, any of the inlets, outlets, and/or vent valves canbe sealed prior to first use. Sealing inlets, outlets, and/or ventvalves can prevent evaporation and/or contamination of fluids disposedor stored within the device. A seal over an inlet, outlet, and/or ventvalve can be pierced, removed, or broken to allow external fluids toenter into the inlet and/or vent valve. As a specific example, in someembodiments, vent valve 24 and inlet 14 can be sealed prior to firstuse, and those seals can be pierced, removed, or broken to allowexternal fluids to enter. In certain embodiments, a vent valve isactuated only after the removal of a cover from a vent valve. Inaddition, outlet 15 (or point 90 or outlet 92) can be sealed prior tofirst use, and pierced, removed, or broken just prior to the applicationof a negative pressure (e.g., a vacuum) or to allow for venting (e.g.,in the case where positive pressure is applied to the inlet).

In one particular embodiment, device 10 can be used for performing animmunoassay for human IgG, and can use sliver enhancement for signalamplification. After delivery of a sample (e.g., fluid 22) containinghuman IgG from channel 12 to the reaction area, binding between thehuman IgG and a stored dry reagent, anti-human IgG, can take place. Thisbinding can form a binding pair complex in a detection zone (e.g.,comprising a detector) proximate the reaction area. Stored reagents fromupstream portions of channel 12 can then flow over this binding paircomplex. One of the stored fluids (e.g., fluid 20) may include asolution of metal colloid (e.g., a gold conjugated antibody) thatspecifically binds to the antigen to be detected (e.g., human IgG). Thismetal colloid can provide a catalytic surface for the deposition of anopaque material, such as a layer of metal (e.g., a multitude of silvergrains), on a surface of the detection zone. The layer of metal can beformed by using a two component system. In some cases, a metal precursor(e.g., a solution of silver salts) can be contained in fluid 62 storedin channel 36, and a reducing agent (e.g., hydroquinone, or otherreducing agent listed above) can be contained in fluid 64 stored inchannel 38. These two components, which can produce signal amplificationupon mixing, are reactive with each others, and can only be maintainedas a mixture for a few minutes. For that reason, they are storedindividually and they cannot mix with each other until the flow drivesboth solutions towards the intersection near vent valve 34. Whennegative pressure is applied to outlet 92, and vent valves 24 and 34 areclosed, the silver salt and hydroquinone solutions eventually merge atthe intersection proximate vent valve 34, where they can mix slowly(e.g., due to diffusion) as they flow along channel 12, and then flowover the reaction area. Therefore, if antibody-antigen binding occurs inthe reaction area, the flowing of the metal precursor solution throughthe area can result in the formation of an opaque layer, such as asilver layer, due to the presence of the catalytic metal colloidassociated with the antibody-antigen complex. The opaque layer mayinclude a substance that interferes with the transmittance of light atone or more wavelengths. Any opaque layer that is formed in themicrofluidic channel can be detected optically, for example, bymeasuring a reduction in light transmittance through a portion of thereaction area (e.g., a meandering channel) compared to a portion of anarea that does not include the antibody or antigen. Alternatively, asignal can be obtained by measuring the variation of light transmittanceas a function of time, as the film is being formed in a detection zone.The opaque layer may provide an increase in assay sensitivity whencompared to techniques that do not form an opaque layer.

Although immunoassays are primarily described, it should be understoodthat devices described herein may be used for any suitable chemicaland/or biological reaction, and may include, for example, othersolid-phase assays that involve affinity reaction between proteins orother biomolecules (e.g., DNA, RNA, carbohydrates), or non-naturallyoccurring molecules (e.g., aptamers, synthetic amino acids).

The flow of fluid within a channel can be achieved by any suitablemethod. In some embodiments, flow is achieved by establishing a pressuregradient within the channel in which the fluid is contained. Such apressure gradient can be established, for example, by applying anegative pressure to one end of a channel (e.g., an outlet of achannel). Exemplary methods of applying negative pressure include, butare not limited to, attachment of a vacuum pump to an outlet, withdrawalof air from a syringe attached to an outlet, or by any other suitablemethod.

A pressure gradient can also be established by applying a positivepressure at one or more vent valves and a relatively smaller pressure,such as ambient pressure, at the outlet. For example, in FIGS. 4A-4C,outlet 202 may be exposed to ambient pressure. Positive pressure aboveambient may be applied through an open vent valve 208, which wouldresult in fluid flow in the direction of the arrows shown in FIG. 4A, aslong as inlets 210 and 214 remained closed. As shown illustratively inFIG. 4B, vent valve 208 can be closed and inlets 210 and 214 opened topressure above ambient. To move a mixed plug of fluid as shown in FIG.4C, inlets 210 and 214 can be closed while 208 is reopened to positivepressure. The use of positive pressure may involve closing all of thevent valves associated with the device, except those on the desired pathof flow. The closure of any vent valve may be fluid tight. Positivepressure can be applied, for example, via a pump, by use of gravity, orany other suitable method.

In certain embodiments, the pressure applied to induce fluid flow (e.g.,a positive or negative pressure) from a fluid flow source (e.g., avacuum or a pump) remains substantially constant during the carrying outof a process (e.g., a reaction) in the device after initial applicationof the fluid flow source to the channel system, even when valves and/orother components described herein are actuated. However, the linear flowrate of fluids in the channel can vary, and may be controlled by variousmethods such as those described in U.S. patent application Ser. No.12/428,372, filed Apr. 22, 2009, entitled “Flow Control in MicrofluidicSystems” and published as U.S. Publication No. 2009/0266421, which isincorporated herein by reference. In other embodiments, the pressurefrom a source of fluid flow can be varied during operation of thedevice.

In some embodiments, a chemical and/or biological reaction involvesbinding. Different types of binding may take place in devices describedherein. The term “binding” refers to the interaction between acorresponding pair of molecules that exhibit mutual affinity or bindingcapacity, typically specific or non-specific binding or interaction,including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

In some cases, a heterogeneous reaction (or assay) may take place in achannel; for example, a binding partner may be associated with a surfaceof a channel, and the complementary binding partner may be present inthe fluid phase. The term “binding partner” refers to a molecule thatcan undergo binding with a particular molecule. Biological bindingpartners are examples; for instance, Protein A is a binding partner ofthe biological molecule IgG, and vice versa. Likewise, an antibody is abinding partner to its antigen, and vice versa. In other cases, ahomogeneous reaction may occur in the channel. For instance, bothbinding partners can be present in the fluid phase (e.g., in two-fluidlaminar flow system). Non-limiting examples of typical reactions thatcan be performed in a meandering channel system include chemicalreactions, enzymatic reactions, immuno-based reactions (e.g.,antigen-antibody), and cell-based reactions.

A device can be fabricated of any material suitable. Non-limitingexamples of materials include polymers (e.g., polyethylene, polystyrene,polycarbonate, poly(dimethylsiloxane), PMMA, PFFE, a cyclo-olefincopolymer (COC), and cyclo-olefin polymer (COP)), glass, quartz, andsilicon. Those of ordinary skill in the art can readily select asuitable material based upon e.g., its rigidity, its inertness to (e.g.,freedom from degradation by) a fluid to be passed through it, itsrobustness at a temperature at which a particular device is to be used,and/or its transparency/opacity to light (e.g., in the ultraviolet andvisible regions). In some embodiments, the material and dimensions(e.g., thickness) of a substrate are chosen such that the substrate issubstantially impermeable to water vapor.

In some instances, a microfluidic substrate is comprised of acombination of two or more materials, such as the ones listed above. Forinstance, the channels of the device may be formed in a first material(e.g., poly(dimethylsiloxane)), and a cover that is formed in a secondmaterial (e.g., polystyrene) may be used to seal the channels. Inanother embodiment, a channels of the device may be formed inpolystyrene or other polymers (e.g., by injection molding) and abiocompatible tape may be used to seal the channels. A variety ofmethods can be used to seal a microfluidic channel or portions of achannel, including but not limited to, the use of adhesives, gluing,bonding, welding (e.g., ultrasonic) or by mechanical methods (e.g.,clamping).

A channel can have any cross-sectional shape (circular, semi-circular,oval, semi-oval, triangular, irregular, square or rectangular, or thelike) and can be covered or uncovered. In embodiments where it iscompletely covered, at least one portion of the channel can have across-section that is completely enclosed, or the entire channel may becompletely enclosed along its entire length with the exception of itsinlet(s) and outlet(s). A channel may also have an aspect ratio (lengthto average cross sectional dimension) of at least 2:1, more typically atleast 3:1, 5:1, or 10:1 or more. An open or partially open channel, ifpresent, may include characteristics that facilitate control over fluidtransport, e.g., structural characteristics (an elongated indentation)and/or physical or chemical characteristics (hydrophobicity vs.hydrophilicity) or other characteristics that can exert a force (e.g., acontaining force) on a fluid. The fluid within the channel may partiallyor completely fill the channel. In some cases where an open channel isused, the fluid may be held within the channel, for example, usingsurface tension (e.g., a concave or convex meniscus).

Though in some embodiments, systems of the invention may bemicrofluidic, in certain embodiments, the invention is not limited tomicrofluidic systems and may relate to other types of fluidic systems.“Microfluidic,” as used herein, refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension of at least 3:1. A “microfluidic channel,” as used herein, isa channel meeting these criteria.

The “cross-sectional dimension” (e.g., a diameter) of the channel ismeasured perpendicular to the direction of fluid flow. Most fluidchannels in components of the invention have maximum cross-sectionaldimensions less than 2 mm, and in some cases, less than 1 mm. In one setof embodiments, all fluid channels containing embodiments of theinvention are microfluidic or have a largest cross sectional dimensionof no more than 2 mm or 1 mm. In another set of embodiments, the maximumcross-sectional dimension of the channel(s) containing embodiments ofthe invention are less than 500 microns, less than 200 microns, lessthan 100 microns, less than 50 microns, or less than 25 microns. In somecases the dimensions of the channel may be chosen such that fluid isable to freely flow through the article or substrate. The dimensions ofthe channel may also be chosen, for example, to allow a certainvolumetric or linear flowrate of fluid in the channel. Of course, thenumber of channels and the shape of the channels can be varied by anymethod known to those of ordinary skill in the art. In some cases, morethan one channel or capillary may be used.

In some instances, a reagent is positioned in a channel prior tocomplete fabrication of a microfluidic channel system. A microfluidicchannel system is not complete if, for example, a system that isdesigned to have enclosed channels has channels that are not yetcompletely enclosed. A channel is enclosed if at least one portion ofthe channel has a cross-section that is completely enclosed, or if theentire channel is completely enclosed along its entire length with theexception of its inlet(s) and/or outlet(s).

Wet reagents are typically stored in a microfluidic system afterchannels of the system have been completely covered. A fluid reagent tobe stored in the system may be introduced into an inlet of a channel,and after at least partially filling the channel with the fluid, theinlet(s) and/or outlet(s) of the channel can be sealed, for example, toretain the fluid and to prevent contamination from external sources.

The term “determining,” as used herein, generally refers to themeasurement and/or analysis of a substance (e.g., within a reactionsite), for example, quantitatively or qualitatively, or the detection ofthe presence or absence of the substance. “Determining” may also referto the measurement and/or analysis of an interaction between two or moresubstances, for example, quantitatively or qualitatively, or bydetecting the presence or absence of the interaction.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used. Determination techniques may includeoptically-based techniques such as light transmission, light absorbance,light scattering, light reflection and visual techniques. Determinationtechniques may also include luminescence techniques such asphotoluminescence (e.g., fluorescence), chemiluminescence,bioluminescence, and/or electrochemiluminescence. Those of ordinaryskill in the art know how to modify microfluidic devices in accordancewith the determination technique used. For instance, for devicesincluding chemiluminescent species used for determination, an opaqueand/or dark background may be preferred. For determination using metalcolloids, a transparent background may be preferred. Furthermore, anysuitable detector may be used with devices described herein. Forexample, simplified optical detectors, as well as conventionalspectrophotometers and optical readers (e.g., 96-well plate readers) canbe used.

EXAMPLES

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

Methods for fabricating a microfluidic channel system are described.

Channel systems, such as the ones shown in FIGS. 1A and 1B, weredesigned with a computer-aided design (CAD) program. The microfluidicdevices were formed in poly(dimethylsiloxane) Sylgard 184 (PDMS, DowCorning, Ellsworth, Germantown, Wis.) by rapid prototyping using mastersmade in SU8 photoresist (MicroChem, Newton, Mass.). The masters wereproduced on a silicon wafer and were used to replicate the negativepattern in PDMS. The masters contained two levels of SU8, one level witha thickness (height) of ˜70 μm defining the channels in the immunoassayarea, and a second thickness (height) of ˜360 μm defining the reagentstorage and waste containment regions. Another master was designed withchannel having a thickness (height) of 33 μm. The masters were silanizedwith (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (ABC-R,Germany). PDMS was mixed according to the manufacturer's instructionsand poured onto the masters. After polymerization (4 hours, 65° C.), thePDMS replica was peeled off the masters and access ports were punchedout of the PDMS using stainless steel tubing with sharpened edges (1.5mm in diameter). To complete the fluidic network, a flat substrate suchas a glass slide, silicon wafer, polystyrene surface, flat slab of PDMS,or an adhesive tape was used as a cover and placed against the PDMSsurface. The cover was held in place either by van der Waals forces, orfixed to the microfluidic device using an adhesive.

In other embodiments, the microfluidic channels were made inpolystyrene, cyclo-olefin-copolymer, or other thermoplastics byinjection molding. This method is known to those of ordinary skill inthe art. The volume of an injection molding cavity can be defined by abottom surface and a top surface separated by a hollow frame whichdetermines the thickness of the molded article. For an article includingchannel features and or other microscale elements on two opposing sidesof the article, the bottom and top surfaces of the molding cavity mayinclude raised features that create the channel features on either sideof the article. For an article including channel features on only oneside of the article, only the top or bottom surface of the moldingcavity includes such features. Thru-holes that pass through the entirethickness of the article can be produced by pins traversing the cavity,embedded in one or more surfaces of the cavity and contacting the otherside. For instance, the pins may extend from only the top surface, onlythe bottom surface, or from both the top and bottom surfaces. When thecavity is filled with pressurized, molten plasitic and then cooled, anarticle is created with channels on one or both sides and holes servingas connectors or inlets and outlets. To complete the fluid network,adhesive tape was applied to the surfaces of the article to seal thechannels.

Example 2

This example describes the control of movement of fluids in microfluidicsystems comprising a single channel that incorporate at least one ventvalve to control the movement of fluid. FIGS. 5A-5B include schematicillustrations of the systems described in this example.

The system shown in FIG. 5A includes a single channel in which an inlet,an outlet, a vent valve were fabricated. This system was fabricated byinjection molding as described in Example 1. The single channel 302 wasconfigured to flow fluid portions 304 and 306 in the direction of arrow308. Water was used for the fluid portions 304 and 306 in thisexperiment, and these fluid portions were separated by a plug of air.The channel included vent valve 310 and inlet 312 upstream of vent valve310. A vacuum operating at a substantially constant pressure of −40 kPawas applied at channel outlet 314 to provide a pressure drop across themicrofluidic channel during the entire experiment.

When vent valve 310 was opened, it functioned as a preferential vent,meaning air flowed through the valve to replace the fluid leaving thesystem through the outlet. Fluids located upstream of vent valve 310(including the fluid between valve 310 and inlet 312) did not flowregardless of whether the inlet was open or closed. When vent valve 310was closed, all of the fluid in the channel flowed as long as inlet 312was open. In this way, vent valve 310 was used to control delivery of afluid in a microfluidic channel. Note that when both vent valve 310 andinlet 312 were closed, no fluid flowed through the channel (althoughsome motion was observed due to fluid expansion when vacuum wasapplied).

The system shown in FIG. 5B includes a single channel in which threevent valves were incorporated. The single channel 320 was configured toflow fluid portions 322, 324, 326, and 328 in the direction of arrow308. The channel included inlet 330 and vent valves 332, 334, and 336.Like the system described in FIG. 5A, a vacuum was applied at channeloutlet 340 to provide a pressure drop across the microfluidic channel.

In one experiment, vent valve 332 was opened and, upon applying thevacuum to outlet 340, only fluid 322 was transported through channel320. Subsequently, vent valve 332 was closed while valve 334 was opened,resulting in the transport of only fluid 324 through channel 320. Next,vent valves 332 and 334 were closed while valve 336 was opened, andfluid portion 326 was transported through the channel. Finally, ventvalves 332, 334, and 336 were closed while inlet 330 was opened,resulting in the transport of fluid portion 328 through the channel.

In another set of experiments, multiple fluids were transported throughthe channel simultaneously. In one case, prior to first use, vent valve332 was closed, but valve 334 was opened. Upon applying vacuum to theoutlet 340, fluid portions 322 and 324 were simultaneously transportedthrough channel 320 in the direction of arrow 308. In anotherexperiment, prior to first use, vent valves 332 and 334 were closed, butvalve 336 was opened. Upon applying vacuum to the outlet 340, fluidportions 322, 324, and 326 were simultaneously transported throughchannel 320 in the direction of arrow 308. Finally, in one experiment,all of the vent valves were closed, and inlet 330 was opened, resultingin the simultaneous transport of fluid portions 322, 324, 326, and 328,upon applying vacuum to the outlet 340.

This example shows that fluid control, including the timing of fluidplugs, can be achieved in a device by opening and closing one or morevent valves and by applying a single source of fluid flow (e.g., avacuum) operated at a substantially constant pressure throughout the useof the device.

Example 3

This example describes the control of movement of fluids in microfluidicsystems comprising multiple channels and at least one vent valve tocontrol the movement of fluid. FIGS. 6A-6C include schematicillustrations of the systems described in this example. In the deviceillustrated in FIG. 6A, a microchannel 410 was fluidically connected totwo channel branches 412 and 414, which intersected at vent valve 416.Microchannel 410 contained fluid 418. In addition, fluids 420 and 422were stored in branches 412 and 414, respectively. Channel 410 wasconnected to outlet 424, while branches 412 and 414 were connected toinlets 426 and 428, respectively. All of the fluids in the device wereseparated by plugs of gas (immiscible with fluid 418, 420 and 422).

A vacuum operating at a substantially constant pressure of −40 kPaduring the entire experiment was attached to outlet 424. Initially, ventvalve 416 was opened, which caused fluid 418 to flow throughmicrochannel 410 in the direction of arrow 408 and air to flow throughvent valve 416. Fluids 420 and 422 did not move even though inlets 426and 428 were open. After fluid 418 exited outlet 424, the flow rate ofthe gas through vent valve 416 increased, due to the elimination of thepressure drop caused by fluid 418. Next, vent valve 416 was closed. Oncethe vent valve was closed, fluids 420 and 422 were mixed at vent valve416 to produce mixed fluid 430 (shown in FIG. 6B).

In another set of experiments, fluids 420 and 422 were transportedsequentially, rather than simultaneously, past vent valve 416. In afirst experiment, in the embodiment illustrated in FIG. 6C, vent valve416 and inlet 426 were both closed (while inlet 428 was opened) afterfluid 418 was transported through outlet 424. By closing inlet 426,fluid 420 was held substantially stationary in branch 412 due to theinability of gas to enter inlet 426. On the other hand, fluid 422 wastransported through branch 414 and past closed vent valve 416 as gas wastransported through inlet 428.

This example shows that fluid control, including mixing and the timingof fluid plugs, can be achieved in a device by opening and closing oneor more vent valves and by applying a single source of fluid flow (e.g.,a vacuum) operated at a substantially constant pressure throughout theuse of the device.

Example 4

This example describes the use of a branched channel system to performan assay in which an optically detectable signal by electrolesslydepositing silver onto gold particles. FIG. 7 includes a schematicillustration of the assay device 300 used in this example. The assayused in this example is generally described in International PatentPublication No. WO2005/066613 (International Patent Application SerialNo. PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Deviceand Method,” which is incorporated herein by reference in its entiretyfor all purposes.

The device included reaction area 510, waste containment region 512, andan outlet 514. The reaction area included a microfluidic channel 50microns deep and 120 microns wide, with a total length of 175 mm. Thedevice also included microfluidic channel 516 and channel branches 518and 520 (with inlets 519 and 521, respectively). Channel 516 andbranches 518 and 520 were 350 microns deep and 500 microns wide. Inaddition, channel 516 was 390 mm long, and branches 518 and 520 wereeach 360 mm long. The reaction area and microfluidic channels werefabricated as described in Example 1. Before sealing the channels,anti-PSA antibodies were attached to a surface of the device in asegment of the reaction area 510.

Prior to first use, the device was loaded with liquid reagents. Thefollowing sequence of liquids were loaded into channel 516: a 2microliter plug of water 542, a 2 microliter plug of buffer solution541, a 20 microliter plug of aqueous solution containing anti-PSAantibodies labeled with colloidal gold 526, a microliter plug of buffersolution 524. This sequence of fluid plugs was loaded using a pipettethrough the inlet port 539. Fluid 528, containing a solution of silversalt, was loaded into branching channel through port 519 using apipette. Fluid 530, containing a reducing solution, was loaded intobranching channel 520 through port 521. Each of the liquids shown inFIG. 7 were separated from the other liquids by plugs of air. Ports 514,519, 521, 536, 539, and 540 were sealed with an adhesive tape that canbe easily removed or pierced. As such, the liquids were stored in thedevice prior to first use.

At first use, the ports 514, 519, 521, 536, 539, and 540 were unsealed.A tube 544 containing 10 microliters of sample blood (522) was connectedto ports 539 and 540. This created a fluidic connection between reactionarea 510 and channel 516, which were otherwise unconnected and not influid communication with one another prior to first use. A vacuum of −40kPa was applied to port 514. Sample 522 was flowed in the direction ofarrow 538 into reaction area 510. As the fluid passed through thereaction area, the PSA proteins in sample 522 were captured by anti-PSAantibodies immobilized on the reaction area walls. The sample took 5minutes to pass through the reaction area, after which it was capturedin the waste containment region 512. Exemplary waste containment regionsthat can be used in devices described herein are provided in U.S. patentapplication Ser. No. 12/196,392, filed Aug. 22, 2008, entitled “Liquidcontainment for integrated assays” and published as U.S. Publication No.2009/0075390, which is incorporated herein by reference.

Fluids 524, 526, 541, and 542 followed the sample through the reactionarea 510 towards waste containment region 512. This resulted in thetransport of fluid 524 in the direction of arrow 538 to reaction area510. As fluid 524 was passed through the reaction area, it washed awayremaining unbound sample components. As fluid 526 passed through thereaction area, gold-labeled anti-PSA antibodies were coupled to the PSAcaptured on the reaction area walls (to form a sandwich immunocomplex).Fluids 541 and 542 followed and further washed the reaction area of anyunbound reagent component. The last wash fluid 542 (water) washed awaysalts that could react with silver salts (i.e., chloride, phosphate,azide).

Silver can be deposited on the captured gold particles to increase thesize of the colloids to amplify the signal. In some embodiments, thesignal can be recorded by optical means as optical density. Toaccomplish this, fluids 528 and 530 were mixed to produce a reactivesilver solution. The ratio of the volumes of fluids 528 and 530 wasabout 1:1. To initiate the mixing of fluids 528 and 530, vent valve 536was closed while the vacuum applied at 514 was maintained, resulting inthe simultaneous flow of fluids 528 and 530 towards vent valve 536. Thevent valve was closed to initiate mixing only after the final previousfluid 542 had exited the reaction area. Closure was performed in oneexperiment by sealing port 536 with an adhesive tape. In anotherexperiment, a tube (not shown) operatively associated with a solenoidvalve (SMC V124A-6G-M5, not shown) was connected to vent valve 536 withan o-ring tight fit. The solenoid valve was activated to seal the port(and later deactivated to unseal the port) in a manner similar to thatdescribed herein with respect to FIGS. 2E-2F. Fluids 528 and 530 mixedwith each other at vent valve 536, producing an activated silversolution with a viscosity of about 1×10⁻³ Pa s. The cross-sectional areaof the microfluidic channel under vent valve 536 was about twice that ofchannels 518 and 520. After 10 seconds, vent valve 536 was opened. Atthat time, approximately 55% of both fluids 528 and 530 had mixed, andthe remaining fluids 528 and 530 were left in channels 518 and 520,respectively.

The activated silver solution was flowed through reaction area 510 toprovide the silver for the deposition. Because the mixed solution isstable for only a few minutes (usually less than 10 minutes) the mixingwas performed less than a minute before use in reaction area 510.Moreover, in order to achieve a reproducible deposition of silver on thecolloids, the time between mixing of the reagents to produce theactivated silver solution and the delivery of the activated silversolution to the reaction area were controlled such that they wereconsistent among multiple experiments.

The control of the flow rates of the fluids within channel 516 and thereaction area 510 were important when flowing fluids through the system.Due to the reaction area's relatively small cross sectional area, itserved as a bottleneck, controlling the overall flow rate in the system.When the reaction area contained liquids, the linear flow rates of thefluids in channel 516 was about 0.5 mm s⁻¹. Fluids flowing frombranching channels 518 and 520 into main channel 516 might not havemixed reproducibly at this rate, as one fluid might have flowed fasterthan the other, causing unequal portions of fluids 528 and 530 to bemixed. On the other hand, when the reaction area contained air, thelinear flow rates of the fluids in channel 516 and branching channels518 and 520 were about 15 mm s⁻¹. At this higher flow rate, the flowrate in branching channels 518 and 520 were equal and reproducible (whenvent valve 536 was closed), producing reproducible mixing. For thisreason, vent valve 536 was not closed until fluid 542 passed through thereaction area to the waste containment region. One could determine whenfluid 542 had exited the reaction area 510 visually (by eye).Alternatively, an optical detector was positioned so as to measuretransmission of light through part of reaction area 510, as described inmore detail in more detail in International Patent Publication No.WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod”, which is incorporated herein by reference.

The microfluidic system shown in FIG. 7 was designed such that thevolume of the channel between vent valve 536 and reaction area 510 waslarger than the expected volume of the mixed activated silver solution(i.e., the combined portion of fluids 528 and 530 which traveled intochannel 516 while vent valve 536 was closed). This ensured thatsubstantially all of the mixing took place at a relatively high linearflow rate (since no liquid, and only air, was present in the reactionarea 510 at this time), and before the activated solution reached thereaction area. This configuration helped promote reproducible and equalmixing.

For the assay described in this example, it was important to sustain aflow of the activated silver mixture within the reaction area for a fewminutes (e.g., 2 to 10 minutes). In a first experiment, 45 microlitervolumes of fluids 528 and 530 were loaded, of which a portion was usedfor mixing (producing a total of 55 microliters of activated silversolution). This volume of combined fluid had a residence time in thereaction area of about 300 seconds. However, the use of this relativelysmall volumes of liquid could pose a challenge. When relatively shortlengths of fluid segments 528 and 530 are used, it can be relativelydifficult to ensure that 1:1 ratios of the two fluids were mixed. Smallvariations in segment length could produce uneven flow rates of the twoliquids, with a shorter segment exhibiting a relatively high flow rate(due to a relatively small resistance to flow and) compared to thelonger segment. This effect can produce a deviation in mixing ratio.

To characterize this effect, a second set of experiments was performedin which a 45 microliter volume of silver salt solution and a 45microliter volume of reducing solution were mixed to produce a 90microliter volume of activated silver solution. The silver salt solutionwas found to flow slightly faster (for a combination of reasonsincluding slight differences in formulation, due to the difference inchemical composition, and slight variation in channel cross section, dueto the tolerances of the machining techniques used for the channelfabrication) relative to the reducing solution, and therefore, exhibiteda slightly faster flow rate through its branch when the vacuum wasapplied. FIG. 8 includes a plot of the volumes of the silver saltsolution (dotted line) and the reducing solution (solid line) that haveentered the mixing channel (in microliters) as a function of the timeelapsed after the initial contact of the silver salt and reducingsolutions. This difference in flow rate is indicated by the slightdifference in the slope of the lines in FIG. 8 from t=0 to t=9 seconds.At t=9 seconds, the absolute difference in lengths of the segmentsbecomes important, and the silver salt solution (having a faster flowrate, and thus a shorter segment of liquid remaining in its branch)flowed even faster relative to the reducing solution. This effect isillustrated by upward trend of the silver salt curve (relative to thelinear extrapolation), and the downward trend of the reducing solutioncurve.

In addition, it was observed that if the trailing edge of one of fluids528 and 530 reagents reached vent valve 536, a slight burst of liquidwas ejected toward the top of the hole in vent valve 536. That liquidwas found to enter into contact with the external valving mechanism.While this had no immediate observable effect on the valving efficiency,it resulted in unwanted contamination of the valve. Repeated use of thevalve in this manner (e.g., to run multiple experiments) might alter thenormal function of the valve. By re-opening vent-valve 536 before all offluids 528 and 530 have mixed ensured that neither of the trailing edgesof fluids 528 and 530 reached vent valve 526, and no liquid ejectionoccurred. Thus, by loading excess reagent into braches 518 and 520 (toensure there are no large variations between the lengths of fluids 528and 530 during flow), and by using no more than about ⅔ of the volume ofthe stored reagent before re-opening vent valve 536, a consistent mixingratio was maintained throughout the mixing step while avoiding liquidprojection/contamination of the external valving mechanism in vent valve536. The valve may be re-opened at various stages of completiondepending of the flow behavior of a specific set of reagents.

This example shows that fluid control, including the mixing of reagents,the changing of flow rates, and the timing of fluid flow can be achievedin a device to perform an assay by opening and closing one or more ventvalves and by applying a single source of fluid flow (e.g., a vacuum)operated at a substantially constant pressure throughout the use of thedevice. This example also shows the importance of controlling flow ratesof individual plugs of fluid to be mixed in a device.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A method, comprising: providing a device comprising: a main channel;a first branching channel containing a first fluid; a second branchingchannel containing a second fluid, wherein the first and secondbranching channels connect at an intersection and are fluidicallyconnected to the main channel; and a vent valve positioned between aportion of the first branching channel and a portion of the mainchannel; actuating the vent valve; causing the first and second fluidsto flow into the intersection substantially simultaneously; and mixingat least portions of the first and second fluids to produce a mixedfluid.
 2. A method, comprising: providing a device comprising: anupstream channel portion containing a first fluid; a downstream channelportion containing a second fluid different from the first fluid; a ventvalve positioned between the upstream and downstream channel portions;while the first and second fluids are in fluid communication with oneanother, flowing the second fluid in the downstream channel portionwithout substantially flowing the first fluid; and flowing the firstfluid from the upstream channel portion to the downstream channelportion after the flowing of the second fluid.
 3. A device, comprising:an inlet; an outlet; an upstream channel portion in fluid communicationwith the inlet; a downstream channel portion in fluid communication withthe outlet; a vent valve positioned between the downstream and upstreamchannel portions; and a first fluid stored in at least one of theupstream and downstream channel portions, wherein the device is sealedand constructed and arranged for storing the first fluid in the devicefor at least one hour prior to first use.
 4. A device, comprising: aninlet; an outlet; a main channel between the inlet and the outlet; and afirst and a second vent valve positioned in series along the mainchannel between the inlet and the outlet.
 5. A method as in claim 1,wherein at least one of the channels is a microfluidic channel. 6.(canceled)
 7. A method as in claim 1, wherein causing the first and/orsecond fluids to flow comprises applying a vacuum to one end of thechannel containing the first fluid and/or second fluids.
 8. A method asin claim 1, wherein the first fluid is a liquid.
 9. (canceled)
 10. Amethod as in claim 1, wherein the second fluid is a liquid. 11-15.(canceled)
 16. A method as in claim 1, comprising storing the firstfluid in the device prior to first use, wherein the first and secondbranching channels are in fluid communication with the main channelduring storage of the first fluid.
 17. (canceled)
 18. A method as inclaim 1, wherein the first fluid comprises a metal solution.
 19. Amethod as in claim 1, wherein the second fluid comprises a reducingagent.
 20. (canceled)
 21. A method as in claim 1, wherein the first andsecond fluids are separated by a third fluid immiscible with both thefirst and second fluids.
 22. A method as in claim 1, wherein the valveis positioned between the first fluid and the second fluid. 23-26.(canceled)
 27. A method as in claim 1, further comprising contacting themixed first and second fluids with a reaction area w/in 10 minutes ofmixing the first and second fluids.
 28. (canceled)
 29. A method as inclaim 1, wherein the intersection of the first and second branchingchannels comprises a mixing region, the mixing region having a largercross-sectional area than either of the first or second branchingchannels.
 30. A method as in claim 29, wherein the mixing regioncomprises a vent valve.
 31. A method as in claim 1, wherein the mixingof at least portions of the first and second fluids includes turbulentmixing.
 32. A method as in claim 1, wherein the main channel issufficiently long to allow for complete mixing of the first and secondfluids via diffusion.
 33. A method as in claim 1, further comprising abinding partner disposed in a reaction area downstream of theintersection.
 34. A method as in claim 1, wherein at least one of thefluids contains a reagent for a chemical and/or biological reaction. 35.A method as in claim 1, wherein the first fluid contains a first reagentfor a chemical and/or biological reaction, and the second fluid containsa second reagent for the chemical and/or biological reaction that isdifferent from the first reagent.
 36. A method as in claim 34, whereinthe reagent participates in a heterogeneous affinity reaction. 37-40.(canceled)
 41. A method as in claim 1, wherein the cross-sectional areasof at least one of the first branching channel and the second branchingchannel are selected such that, when equal pressures are applied to thefirst and second branching channels, the first and second fluids flowinto the intersection substantially simultaneously.
 42. A method as inclaim 1, comprising flowing a third fluid in a main channel beforeactuating the vent valve, without substantially flowing the first andsecond fluids.
 43. A method as in claim 1, wherein a substantiallyconstant vacuum is applied at the outlet of the main channel and timingof the flow of the third, second, and first fluids is accomplished bytiming of actuation of the vent valve.
 44. A method as in claim 1,comprising waiting a predetermined time after actuating the vent valvein order to allow for a predetermined amount of mixing of the at leastportions of the first and second fluids and then opening the vent valveto stop the flow of the first and second fluids remaining in the firstand second branching channels, respectively, thereby delivering apredetermined mixed amount of the first and second fluids to the mainchannel.